Biofilms of Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus epidermidis, Streptococcus pyogenes, and Candida albicans, established in the wells of a polyethylene contact lens case, retained viability to certain soft contact lens disinfectant solutions after exposure for the manufacturer's minimum recommended disinfection times. The relative order of resistance of bacterial biofilms was as follows: S marcescens was greater than P aeruginosa, which was greater than S epidermidis, which was greater than S pyogenes. Air drying of biofilms for 10 hours increased the efficacy of the disinfectant solutions, but drying was not enough to decrease the incidence of recovery to 0% for all solutions. Hydrogen peroxide was more effective against biofilms than disinfectant solutions formulated with chlorhexidine gluconate or polyquaternium-1 or polyaminopropyl biguanide. We recommend that determination of efficacy of contact lens disinfectant solutions should include challenges against biofilms.